Caffeine Detection Via Internally-Referenced Two Part Assay

ABSTRACT

Methods, compositions, and apparatus for detecting the presence of caffeine in a liquid sample are provided. In certain embodiments, an internally referenced competitive assay allows a very precise determination of a threshold value of caffeine for use in semiquantitative types of ligand-receptor assays. By using a detection means that participates in two assays, sensitivity is doubled in the maximum sensitivity range and the range can be adjusted to match the predicted concentration range of an analyte. This format and the materials described herein allow the assay to complete within three minutes. In addition, this format accommodates common attributes of liquid samples for detecting caffeine, such as the inclusion of milk or sugar in a coffee-type beverage.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/530,232, filed Sep. 8, 2006, which is incorporated by reference inits entirety.

BACKGROUND

1. Field of the Invention

The invention relates generally to methods, apparatus and compositions,useful for detecting the presence of caffeine in a liquid sample, andmore specifically, for detecting the presence of caffeine in a beverage.

2. Description of the Related Art

Competitive ligand-receptor assays. Ligand-receptor assays takeadvantage of the ability of bioreagents to identify and quantify minuteamounts of a wide range of substances, also referred to as analytes,with a high degree of specificity and sensitivity. Competitiveligand-receptor assays are one variant of ligand-receptor assays ingeneral. In competitive ligand-receptor assays, analyte substances inthe sample compete with another substance, for example asignal-producing substance, for a limited number of binding sites on thecounterpart of the ligand-receptor pair. After the binding has takenplace, the amount of other substance bound to the counterpart isdetected by any of several means. The signal intensity of competitiveligand-receptor assay is in an inverse relationship with theconcentration of analyte present; thus, a sample with no analyte willgive a maximum signal intensity, and a sample with a range of analyteconcentration will produce less than a maximum signal. Thus,conventional competitive ligand-receptor assays, acting alone, have amaximum sensitivity in a narrow analyte concentration range and requireexternal calibration and standardization.

An additional disadvantage of traditional competitive ligand-receptorassays is that they require external calibration. This disadvantagemanifests itself in so-called semi-quantitative assays, where a “yes” or“no” is indicated by the assay based on the presence or absence of apredetermined concentration of analyte. Such semi-quantitativecompetitive ligand-receptor assays are difficult to perform withoutexternal calibration, thus limiting their usefulness in a variety ofimportant market segments. Due to the inverse relationship betweensignal intensity and analyte concentration, all but the mostconcentrated samples will give a signal in the assay, and therefore astandard curve (or at least one control point with a known standard)must be run in parallel with the sample assay to interpret accuratelyany reading of the sample assay. For example, an optical density readingof 0.5 in a competitive immunoassay using enzymes as a signal producingsystem is meaningless. However, if the user runs a known standard of,for example, 10 micrograms per milliliter of analyte and obtains areading of 1.0, then the sample with the reading of 0.5 can be said tobe more concentrated than the 10 microgram per milliliter sample. Theneed for standardization has severely limited the practical usefulnessof current competitive ligand-receptor assays by requiring several runsof the assay to determine one sample concentration. One of the majordisadvantages of the requirement for outside calibration is theconcomitant reduction of precision and accuracy of each assay due tointer-assay variability in the calibration process. Furthermore, whilethere are commercially-available immunochromatographic test stripversions of the ligand-receptor competitive assay available that do notrequire external calibration, these assays are designed to give apositive indication for the analyte at the least sensitive portion ofthe analyte concentration versus signal intensity curve. Thus, theseimmunochromatographic test strips are to be interpreted as positive foranalyte in the sample when no signal is seen at the test line. As oneskilled in the art would recognize, the precision of the determinationof analyte concentration is compromised in such an assay.

Immunoassays. One flavor of ligand-receptor assay is an immunoassay.Various known formats exist for immunoassays, includingimmunochromatographic test strips for detecting small molecule analytes.One format uses a competitive immunoassay, for which the result isrevealed as two lines (negative result) or one line (positive result).Another format displays a single line as an indication of a positiveresult. Drawbacks of these formats include a very low dose-responseratio at the positive/negative cutoff concentration for some analytes,multiplicity of necessary reagents, high cost of production, anduncertain adaptability to the concentration range of interest for someanalytes.

Caffeine detection assays. Immunoassays can be used to detect variousanalytes, including assays using anti-caffeine antibodies to detect thepresence of caffeine. Existing clinical laboratory analyses and teststrip formats using traditional immunoassay techniques for caffeineprovide varying results. In the laboratory setting, the scientificliterature includes methods such as electrometric determination in whicha caffeine-specific electrode is prepared from acaffeine-picrylsulfonate ion-pair complex dissolved in octanol;fluorimetric determination in which a buffered solution of caffeine isoxidized with N-bromosuccinimide and then reacted with dimethylo-phenylenediamine followed by a fluorescence measurement at 480 nm;colormetric determination in which an ethenolic solution of caffeine isoxidized by potassium bromate, dried and then redissolved indimethylformamide followed by an absorbance measurement at 500 nm;Fourier Transform Infrared Spectrophotometry (FTIR); thin-layer/gaschromatography; enzyme-linked immunosorbent assays (ELISA) in which acaffeine-containing sample of plasma or serum is dissolved in a bufferedsolution and incubated in a vessel where it competes withperoxidase-labeled caffeine for the binding sites on caffeine antibodiesfollowed by detection of a visible color change with the addition ofo-phenylenediamine; immunoassay of theophylline with cross-sensitivityfor caffeine; and immunoliposome assay of theophylline withcross-sensitivity for caffeine.

There are a number of commercially available lateral-flow type testsdisclosing methods for the detection of large or small analytes, usingeither “typical” competitive inhibition to produce negative or indirectreporting results, i.e., reduction of signal with increasing analyteconcentration, or producing positive or direct reporting results, i.e.,increase in signal with increasing analyte concentration. For example,U.S. Pat. Nos. 5,229,073; 5,591,645; 4,168,146; 4,366,241; 4,855,240;4,861,711; 5,120,643; 4,703,017; 5,451,504; 5,451,507; 5,798,273;6,001,658; and 6,699,722. However, these types of commercially availablelateral-flow type tests use either “typical” competitive inhibition toproduce negative or indirect reporting results, or produce positive ordirect reporting results, which share the drawbacks described above.Available tests also may take too long to produce a result to be viable.

It is relatively easy to determine the presence of a wide variety ofcompounds using analytical chemistry techniques. However, such methodsoften are not available, or practical, for individual consumers seekingto determine the presence or absence of certain compounds in their foodand beverages. For example, although “decaffeinated” coffees, teas, andsoft drinks have become increasingly popular, the average consumer hasno way of verifying the absence (or presence) of caffeine in suchbeverages when receiving them in restaurants and other public andprivate settings.

The rise in consumption of decaffeinated beverages has resulted in partfrom the health concerns of ingesting excessive amounts of caffeine.Caffeine is a bitter crystalline alkaloid. There are a variety ofbiological effects and symptoms caused by the ingestion of caffeineincluding tachycardia, diuresis, headaches, decrease in fine motorcoordination, insomnia, and central neurological stimulation. Excessiveamounts of caffeine can make people tense, irritable, and, in somecases, elevate the heart rate to unsafe levels. Caffeine can alsoirritate the alimentary canal. It is common for people diagnosed withsensitive stomachs and colons, as well as other medical conditions, tobe required to refrain from ingesting caffeine as part of their medicaltreatment. Pregnant women may not drink any caffeinated beverages forfear of a teratogenic effect. Both men and women avoid caffeinatedbeverages because caffeine is a known diuretic. Also, as people age,they become increasingly sensitive to the effects of caffeine. However,an individual requesting a decaffeinated beverage can not be fullycertain of the reduced level or absence of caffeine in the beverage.

The present invention addresses these and other deficiencies of theprior art as described more fully below.

SUMMARY OF THE INVENTION

The present invention is defined by the following claims, and nothing inthis section should be taken as a limitation on those claims. Disclosedherein are methods, compositions, and apparatus for detecting thepresence of caffeine, in a liquid sample.

An internally referenced competitive assay such as described hereineliminates the above-referenced drawbacks. This dual-assay formatprovides for adjustment of the test for the concentration range ofinterest, and the internal calibration feature allows a very precisedetermination of a threshold value of caffeine for use insemiquantitative types of ligand-receptor assays. In addition, the dualassay eliminates the need for external calibration, while retaining thespecificity and sensitivity of traditional competitive assays. By usinga detection means that participates in two assays, sensitivity isdoubled in the maximum sensitivity range and the range can be adjustedto match the predicted concentration range of caffeine. This format andthe materials described herein allow the test to complete within threeminutes. In addition, this format accommodates common attributes ofliquid samples for detecting caffeine, such as the inclusion of milk orsugar in a coffee-type beverage.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, and accompanying drawings, where:

FIG. 1A (“FIG. 1A”) illustrates a testing substrate with a liquid samplebeing applied according to one embodiment of the present invention.

FIGS. 1B and 1C show the flow of the liquid sample across the testingsubstrate of FIG. 1A over time according to one embodiment of thepresent invention.

FIG. 1D illustrates a signal on the testing substrate of FIG. 1 aftercompletion of an assay according to one embodiment of the presentinvention.

FIGS. 2A-2D and 3A-3C illustrate the method of FIGS. 1A-1D in greaterdetail according to one embodiment of the present invention.

FIG. 4A illustrates a testing substrate with a signal representing avery high caffeine level according to one embodiment of the presentinvention.

FIG. 4B illustrates a testing substrate with a signal representing avery low caffeine level according to one embodiment of the presentinvention.

FIG. 4C illustrates a testing substrate with a signal indicating that acaffeine threshold has been exceeded according to one embodiment of thepresent invention.

FIG. 4D illustrates a testing substrate with a signal indicating that acaffeine threshold has not been exceeded according to one embodiment ofthe present invention.

FIG. 5A illustrates a side view of a testing substrate according to oneembodiment of the present invention.

FIG. 5B illustrates a top view of a testing substrate according to oneembodiment of the present invention.

FIGS. 6A-6D illustrate examples of housings for the testing substrateaccording to various embodiments of the present invention.

FIG. 7 illustrates the relationship between the two signals of themethod of the present invention according to one embodiment.

FIG. 8 is a graph showing the cross-reactivity of an antibody accordingto one embodiment of the present invention.

FIGS. 9A and 9B illustrate the IC50 of dose-response curves for fourantibodies according to one embodiment of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Briefly, and as described in more detail below, described herein aremethods, compositions and apparatus for detecting the presence ofcaffeine, in a liquid sample. The present invention provides acompetitive assay for determining whether caffeine is present in aliquid sample. The method includes applying the liquid sample to atesting substrate and detecting a visible signal indicative of the levelof caffeine present in the sample. The assay uses a ligand-receptormethod that eliminates the need for external calibration to obtain anaccurate result, as described in U.S. Pat. Nos. 6,649,418, 6,358,875,6,287,875 and 6,103,536 owned by Silver Lake Research Corporation ofMonrovia, Calif., and each of which are hereby incorporated in theirentirety by reference. Thus, the present invention has a detection meansthat participates in two assays, with the signal intensities of bothassays being related to the concentration of caffeine to be determined.The signal intensity of one of the assays is inversely related to theconcentration of caffeine, and the signal intensity of the second assayis directly related to the concentration of caffeine. The combined assayproceeds quickly, providing the visible signal within three minutes. Inone embodiment, the ligand-receptor pair includes an anti-caffeineantibody, a caffeine analog, and caffeine if present in the liquidsample, e.g., a beverage. In this example, the assay provides at leastan accurate threshold determination of whether the sample iscaffeinated.

DEFINITIONS

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

An “analog” of caffeine is a molecule that binds to the one member of aligand-receptor pair in approximately the same specificity as caffeineitself.

“Bibulous” materials are materials that have the capability to effect achromatographic separation of the contained materials, including paper,nitrocellulose, nylon and the like.

“Direct ligand-receptor pair” refers to the pair that does not containcaffeine or a caffeine analog. This pair is so named because the signalintensity generated by this pair is directly proportional to theconcentration of caffeine in the sample.

The “first member” of ligand-receptor pair refers to the member bound,covalently or non-covalently, at least to a sub-population of theparticles.

“Inverse ligand-receptor pair” refers to the pair of which caffeine, ora caffeine analog, is a member. This pair is so named because the signalintensity generated by this pair is inversely proportional to theconcentration of caffeine in the sample.

“Ligand-receptor pair” refers to compounds having spatial and/or polarfeatures which permit them to bind specifically to each other. Examplesof ligand-receptor pairs useful in the present invention includespecific binding pairs such as antigens and antibodies, or fragments ofantibodies, both polyclonal and monoclonal. Members of ligand-receptorpairs may be “engineered,” that is, made by synthetic means.

“Liquid Sample” means liquids or extracts suspected of containingcaffeine.

“Non-bibulous” lateral flow means liquid flow in which all of thedissolved or dispersed components of the liquid are carried atsubstantially equal rates and with relatively unimpaired flow laterallythrough the membrane, as opposed to preferential retention of one ormore components as would occur, for example, in materials capable ofadsorbing or “imbibing” one or more components.

“Non-diffusively bound” means the second members of the inverse anddirect ligand-receptor pairs are either covalently or non-covalentlyattached to the solid support such that advancing liquid does not causeeither member of the pair to substantially move from the place it isapplied on the solid support.

“Particles” can be a wide range of materials known in the art. At leastone sub-population of these particles is composed of a first member of aligand-receptor pair and a signal means, as discussed below. Thus, suchparticles can include enzymes such as glucose oxidase, horseradishperoxidase, alkaline phosphatase, galactosidase, or oxidoreductase. Suchan enzyme, along with its signal producing system, such as described inPawlak et al., International Patent Application No. WO 95/01775; acarbon sol, such as discussed in U.S. Pat. No. 5,559,041; erythrocyteghosts, liposomes, and colored latex particles, such as discussed inU.S. Pat. Nos. 4,703,017 and 5,252,459; colloidal metal particles, suchas colloidal gold, colloidal silver, colloidal platinum and colloidalselenium, such as discussed in U.S. Pat. Nos. 4,313,734, 4,775,636, and4,954,452; each of these references are incorporated by reference hereinin their entirety. Colorable particles and colorable latex particles arealso known in the art and useful as particles herein, such as discussedin U.S. Pat. Nos. 4,373,932 and 4,837,168, both of which areincorporated herein by reference.

The “second member” of a ligand-receptor pair refers to thecorresponding binding member of the pair non-diffusively bound in asignal ratio area.

“Signal means” refers to any of the conventional signaling methods knownin the art detectable by methods such as visible inspection, UV andvisible spectrophotometry, fluorimetry and radiation counters. In oneembodiment, the signal means can be a property of the particlesthemselves. Alternatively, the signal means may be an inducible propertyof the particles, such as colorable latex particles, such a shown inU.S. Pat. Nos. 4,373,932 and 4,837,168, each incorporated by referenceabove. Alternatively the signal means can be attached, either covalentlyor non-covalently, to either the particle itself, to one or more membersof the ligand-receptor pair bound to the particle, or both.Chemiluminescent molecules, such as luminol, luciferin, lucigenin, oroxalyl chloride can be used as a signal means, for example as describedin U.S. Pat. No. 4,104,029, hereby incorporated by reference herein inits entirety for all purposes. Finally, enzymic systems that react witha colorless substrate to give a colored product, such as horseradishperoxidase and aminoethylcarbazole are useful as signal means.

A “testing substrate” is made of a porous material that is generallyhydrophilic or capable of being rendered hydrophilic, includinginorganic powders such as silica, magnesium sulfate, and alumina;natural polymeric materials such as cotton, particularly cellulosicmaterials and materials derived from cellulose, such as fiber containingpapers, e.g., filter paper, chromatographic paper, etc.; synthetic ormodified naturally occurring polymers, such a nitrocellulose, celluloseacetate, fiberglass, poly(vinyl chloride), polyacrylamide, cross-linkeddextran, agarose, polyacrylate, etc.; either used by themselves or inconjunction with other materials; ceramic materials; and the like.Alternatively, the testing substrate of the present invention isfashioned from non-bibulous lateral flow material. The internallyreferenced assay format described herein is based on the disclosure ofU.S. Pat. Nos. 6,649,418, 6,358,875, 6,287,875 and 6,103,536.Preferably, the testing substrate materials of the present invention arechosen that allow the assay to complete within three minutes ofapplication of the liquid sample.

METHODS AND APPARATUS OF THE INVENTION Method

Competitive ligand-receptor assays. Ligand-receptor assays takeadvantage of the ability of bioreagents to identify and quantify minuteamounts of caffeine, with a high degree of specificity and sensitivity.Competitive ligand-receptor assays are one variant of ligand-receptorassays in general. In competitive ligand-receptor assays, caffeine inthe sample competes with another substance, for example asignal-producing substance, for a limited number of binding sites on thecounterpart of the ligand-receptor pair. After the binding has takenplace, the amount of other substance bound to the counterpart isdetected by any of several means. The signal intensity of competitiveligand-receptor assay is in an inverse relationship with theconcentration of caffeine present; thus, a sample with no caffeine willgive maximum signal intensity, and a sample with a range of caffeineconcentration will produce less than a maximum signal. Thus,conventional competitive ligand-receptor assays, acting alone, have amaximum sensitivity in a narrow concentration range and require externalcalibration and standardization. An internally referenced competitiveassay such as described herein and in U.S. Pat. Nos. 6,649,418,6,358,875, 6,287,875 and 6,103,536, eliminates the need for externalcalibration, while retaining the specificity and sensitivity oftraditional competitive assays. By using a detection means thatparticipates in two assays, sensitivity is doubled in the maximumsensitivity range and the range can be adjusted to match the predictedconcentration range of caffeine.

Immunoassays. One variant of a ligand-receptor assay is an immunoassay.Various known formats exist for immunoassays, includingimmunochromatographic test strips. Included in the scope of theinvention is an internally referenced competitive assay, which is basedon the disclosure of U.S. Pat. Nos. 6,649,418, 6,358,875, 6,287,875 and6,103,536. This dual-assay format provides for adjustment of the testfor the concentration range of interest, and the internal calibrationfeature allows a very precise determination of a threshold value ofcaffeine for use in semiquantitative types of ligand-receptor assays.

Caffeine detection assays. Immunoassays can be used to detect caffeineusing anti-caffeine antibodies to detect the presence of caffeine. Adual, internally referenced assay (based on the disclosure of U.S. Pat.Nos. 6,649,418, 6,358,875, 6,287,875 and 6,103,536) is described herein,which in the present invention allows testing to complete within threeminutes. In addition, this format accommodates common attributes ofliquid samples for detecting caffeine, such as the inclusion of milk orsugar in a coffee-type beverage.

Referring now to FIGS. 1A-D, they illustrate a method 102-104 fordetecting the presence of caffeine in a liquid sample according to oneembodiment. In one embodiment the liquid sample is a beverage served as“decaffeinated.”

FIG. 1A shows a liquid sample 105 being applied 102 to a contact region115 of a testing substrate 110. Various application methods may be usedfor this step. According to one embodiment, the liquid sample 105 isapplied to the testing substrate 110, for example using a pipette ordropper containing the liquid sample 105. The contact region 115 isdipped into the liquid sample 105 according to another embodiment. Ahousing is used to apply the liquid sample 105 to the testing substrate110 according to yet another embodiment, in which the testing substrate110 can be at least partially contained within the housing. Thearchitecture and materials of the testing substrate 110 are describedbelow in conjunction with FIGS. 2A-3C and 5A-5B. Examples of housingarchitecture are shown in FIGS. 6A-6D.

Referring back to FIG. 1, the liquid sample 105 flows 103 towards asignal region 120 of the testing substrate 110. The signal region 120 isspatially distinct from the contact region 115 according to oneembodiment. FIGS. 1B and 1C show the flow 103 over time, as representedby a wavy line 125. A signal 130 then may be detected 104 in the signalregion 120 of the testing substrate 110, as shown in FIG. 1D. Varioussignaling mechanisms may be used, as described below. In a preferredembodiment, the signal is visibly detectable. In other embodiments,detection of a signal and signal strength is accomplished by variousmethods known in the art, as described herein.

FIGS. 2A-2D and 3A-3C illustrate the mechanism for the method of FIGS.1A-1D according to one embodiment. Many aspects of FIGS. 2A-2D and 3A-3Care not shown to scale; rather, portions are enlarged to show detail.

FIG. 2A shows application of a liquid sample 105 to a contact region 115of a testing substrate 110. This example corresponds to a very highlevel of caffeine in the sample. The testing substrate 110 also includesa particle region 122 and a signal region 125. In this example, thesignal region 125 includes a first signal area 205 and a second signalarea 210.

The particle region 122 further includes particles 215 diffusely boundto the testing substrate 110 as described herein. The particle 215materials and respective qualities are further described in conjunctionwith FIGS. 5A-5B. A particle 215 also is shown further enlarged forclarity. The particle 215 includes a first member of an inverseligand-receptor pair 220 (“indirect first member”) and a first member ofa direct ligand-receptor pair 225 (“direct first member”) attached tothe particle 215 surface according to one embodiment. Methods ofattaching the first members 220, 225 to the particle 215 surface arediscussed in greater detail below. The indirect first member 220 is ananti-caffeine antibody and the direct first member 225 is a differentantibody selected such that is does not cross-react with caffeine in theexample shown in FIGS. 2A-2D. In other embodiments, the indirect anddirect first members 220, 225 can be any one member of an inverseligand-receptor pair and one member of a direct ligand-receptor pair,respectively, as described herein. The particle 215 also includes asignal means as described herein.

The first signal area 205 further includes a second member of theinverse ligand-receptor pair 230 (“indirect second member”)non-diffusely bound to the testing substrate 110. The second signal area210 includes a second member of the direct ligand-receptor pair 235(“direct second member”) bound to the testing substrate 110. Methods andtypes of bond involved in the second member-testing substrate bond aredescribed in greater detail in conjunction with FIGS. 5A-5B. Theindirect second member 230 may be caffeine or a caffeine analog; what isimportant is that it acts as a competitive inhibitor of caffeine bindingto indirect first member 220. The direct second member 235 is caffeineor a caffeine analog corresponding to the direct first member 225,which, in preferred embodiments is an antibody, as shown in FIGS. 2A-2D.In other embodiments, the indirect and direct second members 230, 235can be the other member of the inverse ligand-receptor pair and theother member of the direct ligand-receptor pair, respectively, asdescribed herein.

Following application of the liquid sample 105 to the contact region115, if the liquid sample 105 includes caffeine 250, as the sample 105shown in FIG. 2A does, as the liquid flows across the testing substrate110, the caffeine 250 occupies indirect first member 220 receptor sitesas shown in FIG. 2B. As a result, the indirect first member 220 receptorsites are competitively inhibited from binding to the caffeine orcaffeine analog 230, and the particle 215 continues past the firstsignal area 205 as shown in FIG. 2C. As the particle 215 reaches thesecond signal area 210, the direct first member 225 has open receptorsites, that are available for binding to the direct second member 235.Because the direct second member 235 is non-diffusely bound to thetesting substrate 110, the particle 215 becomes immobilized when thedirect second member 235 binds direct first member 225. An illustrationof a testing substrate 110 with a signal corresponding to this exampleis shown in FIG. 4A. The first signal area 205 shows no signal, and thesecond signal area 210 shows a strong signal, indicative of a highcaffeine level.

FIG. 3A shows application of a liquid sample 105 to a contact region 115of a testing substrate 110 similar to FIG. 2A. However, the sample 105shown in this example has no caffeine. As the liquid flows across thetesting substrate 110, no caffeine is available in the liquid sample 105to occupy indirect first member 220 receptor sites. As a result, theindirect first member 220 receptor sites become occupied by 230 (i.e., acompetitive inhibitor of caffeine for first member 220 receptor sites),and the particle 215 is immobilized in the first signal area 205 asshown in FIG. 3C As a result, the particle 215 does not continue intothe second signal area 210. An illustration of a testing substrate 110with a signal corresponding to this example is shown in FIG. 4B. Thefirst signal area 205 shows a strong signal, and the second signal area210 shows no signal, indicative of a very low caffeine level.

FIGS. 4A and 4B illustrate the two extremes of a very high amount ofcaffeine in a sample and a very low amount, or no, caffeine in a sample,respectively. However, there may be present in a liquid such as abeverage, a range of caffeine concentrations between concentrations thatproduce these two signaling extremes. FIGS. 4C and 4D illustrate twoexamples of other caffeine levels, in which some caffeine is present.The more caffeine present, the greater the relative number of particles215 that bind in the second signal area 210, and the higher the signalin the second signal area 210. More caffeine also corresponds torelatively fewer particles 215 that bind in the first signal area 205,and to a lower the signal in the first signal area 205. Thus, comparingFIGS. 4C and 4D, FIG. 4C has the higher concentration level. In oneembodiment, the maximum sensitivity range of the assay can be set suchthat the visible signal indicates whether a caffeine threshold, e.g., 15mg/8 oz, has been met or exceeded. For example, FIG. 4C is an example ofa signal indicating that the threshold has been exceeded and 4D is anexample of a signal indicating that the threshold has not been exceeded.In this example, the test could be articulated that a liquid sample iscaffeinated if the second signal area 210 is darker than the firstsignal region 205.

As the signal generated in the two signal areas is related to theconcentration of caffeine, the comparison will give an accuraterendering of at least a threshold, pre-determined concentration ofcaffeine. The relationship between the two signals is illustrated inFIG. 7. Thus, the possibilities of signal intensity ratios between thetwo areas set forth in Table 1 can be imagined.

TABLE 1 Ratio 1st Signal/ Number First Signal Area Second Signal Area2nd Signal 1 No signal Detectable signal 1/∞ 2 Equal signal Equal signal1 3 Some detectable signal No detectable signal ∞ 4 Detectable, lessthan Detectable signal, more <1 second signal area than first signalarea 5 Detectable signal, more Detectable, less than >1 than secondsignal area first signal area

Entries 1, 3, 4, and 5 in the above Table 1 are represented in FIGS. 4A,4B, 4C, and 4D, respectively.

Combinations of the above possible ratios can be used to determine morethan one predetermined concentration of caffeine in a sample. Thus, onecan control the properties of the assay components (by, e.g., selectingthe relative amounts and affinities of the first and second direct andinverse ligand pair components) such that no signal in the first signalarea and some in the second signal area is indicative of a firstpredetermined concentration threshold or range of caffeine. An equalamount of signal in both areas indicates a second predeterminedconcentration threshold or range and detectable signal in the first areaand no detectable signal in the second area corresponds to a thirdpredetermined concentration threshold or range. Any one or more of theabove possible outcomes can be imagined to give such a multiple caffeineconcentration reading.

Furthermore, the population of particles in the particle region can bemanipulated to shift the range of maximum sensitivity for caffeine. Thusthe population may further comprise a subpopulation of particlescontaining only one of the two first members of either the inverseligand-receptor pair or the direct ligand-receptor pair, in addition tothe signaling means. By this method, the signal intensity in one or theother area can be intensified independent of the concentration ofcaffeine.

Device Architecture

An example of a device for use in conjunction with the methods describedherein is represented schematically in FIGS. 5A & 5B. FIG. 5Aillustrates a side view of a testing substrate 110 according to oneembodiment. The testing substrate 110 includes a particle region 122that is part of a larger sample application zone 505, as part of anoptional application pad 510. These two areas are in fluid communicationwith the one or more signal areas 205, 210 contained on wicking material515, which in turn are in fluid communication with an optional absorbentreservoir 520. The particle region 122, optional application pad 510,sample application zone 505, wicking material 515, the signal area(s)205, 210 and the optional absorbent reservoir 520 comprises the testingsubstrate 110. In FIGS. 5A and 5B, all members of the testing substrate110 are shown mounted in the optional backing material 525. FIG. 5Bshows the particles 215 containing the first members and the signalmeans, are shown in the particle region 122 as they exist beforeapplication of the liquid sample. That is, they are shown as diffusivelybound in an absorbent, non-particle-binding pad 510. The liquid sampleis applied to the sample application zone 505 and is wicked through thesignal areas 205, 210, carrying with it the particles 215. Any excesssample that is not absorbed by the rest of the testing substrate 110along the way is eventually absorbed by the optional absorbent reservoir525.

The two signal areas 205, 210 are placed far enough from the particleregion 122 to allow sufficient binding of caffeine to the first memberof the inverse ligand-receptor pair on the particle before it reachesthe signal area according to one embodiment. The two signal areas are inspatial proximity as mentioned above and are close enough such thatdetection of each individual signal and comparison of the two signals isfacilitated. It is important that the particles and the sample solutionsuspected of containing caffeine first go through the first signal zonebefore entering the second signal zone according to one embodiment.Furthermore, it is necessary that the amount of each second member boundto the solid support is greater than the total amount of thecorresponding first member of a ligand-receptor pair in the populationof particles for caffeine according to one embodiment. It is alsoimportant that no member of any ligand-receptor pair cross-react withany other member of a ligand-receptor pair, be it inverse or direct, orwith any non-caffeine component of the liquid sample according to oneembodiment. As mentioned above, the ratio of the two signal areas can bedetermined by visual inspection, by spectrophotometric means, or byradiation counters, or by any other method known in the art.

The testing substrate 110 referred to herein has at least two, andpreferably three, zones in fluid communication with one another. Thus,as stated above, there is a sample application zone 505, which can bethe same as the particle region 122, and a least one signal area 205,210, all of which are in fluid communication with each other. It ispreferred according to one embodiment that the sample application zone505 and the particle region 122 be separate areas. These areas and theone or more signal areas 205, 210 are arranged as discussed above, andare all spatially distinct. The three areas can be arranged as a stripas shown in FIGS. 2A through 5B. The areas all can be on one piece oftesting substrate, or one or more pieces support containing one or moreareas, or areas can be abutted together on a suitable backing, orotherwise be made in fluid communication with each other on saidbacking. Thus, individual pieces of the areas can adhere to a backing bydouble-sided scotch tape. The backing can be, for example, plasticribbon. See, for example, U.S. Pat. Nos. 5,591,645 and 4,861,711 andEuropean Patent Publication No. 291 194 B1, each of which are herebyincorporated by reference herein in their entirety.

The shape of the solid support can be that of longitudinal strips, aseries of parallel strips, or that of a circular configuration, whereinthe circular configuration can optionally be divided into varioussections. For the latter configuration, see U.S. Pat. No. 5,141,875,incorporated by reference herein. All that is required is aconfiguration for which the areas are arranged as discussed above, andthat the sample is able to traverse them in the order discussed. Thus,for the detection of more than one analyte, a strip with a sampleapplication zone, a particle region, and more than one signal areas, arearranged sequentially along the length of the strip, or along a radiusof a non-linear configuration.

Alternatively, for each analyte, a separate series of particle regionsand signal areas can be placed in parallel on one comparatively widestrip. For a circular conformation, the sample application zone would beplaced at the center of the circle. In concentric rings radiating fromthe center, first would be the particle region and then the one or moresignal areas. Alternatively, a common sample application zone could beused to supply the sample solution to two or more partitioned areas ofthe circular device containing separate particle regions and one or moresignal areas.

The testing substrate may be porous material having pores of at least0.1 μm, preferably at least 1.0 mμ, which is susceptible to traversal byan aqueous medium in response to capillary force. Such materials aregenerally hydrophilic or are capable of being rendered hydrophilic andinclude inorganic powders such as silica, magnesium sulfate, andalumina; natural polymeric materials such as cotton, particularlycellulosic materials and materials derived from cellulose, such as fibercontaining papers, e.g., filter paper, chromatographic paper, etc.;synthetic or modified naturally occurring polymers, such anitrocellulose, cellulose acetate, fiberglass, poly(vinyl chloride),polyacrylamide, cross-linked dextran, agarose, polyacrylate, etc.;either used by themselves or in conjunction with other materials;ceramic materials; and the like. The testing substrate should notinterfere with the signal means. This porous material can be attached torigid or semi-rigid backing. On the other hand, the porous material mayprovide its own support. The porous material may be polyfunctional or becapable of being polyfunctionalized to permit covalent bonding ofmembers of a ligand-receptor pair, as well as to permit bonding of anyother components that are part of the device.

Further examples of the porous testing substrate of the presentinvention may be found in assays described, for example, in U.S. Pat.Nos. 4,861,711 and 5,591,645, European Patent Publication No. 291,194and 323,605, each of which is incorporated herein by reference.

Alternatively, the testing substrate of the present invention isfashioned from non-bibulous lateral flow material. By “non-bibulous”lateral flow is meant liquid flow in which all of the dissolved ordispersed components of the liquid are carried at substantially equalrates and with relatively unimpaired flow laterally through themembrane, as opposed to preferential retention of one or more componentsas would occur, for example, in materials capable of adsorbing or“imbibing” one or more components. “Bibulous” materials include paper,nitrocellulose, nylon and the like, which have the capability to effecta chromatographic separation of the contained materials.

An example of the non-bibulous testing substrate material in whichcapillary, non-bibulous lateral flow occurs is glass fiber filter,manufactured by a number of suppliers including Whatman PLC ofMiddlesex, UK. This material has a typical thickness of 0.1-1 mm adensity of 25-800 g/m², and a flow rate of <100 sec/5 cm. There are manyother types of materials that have been used for capillary non-bibulouslateral flow, including cellulose, surface-modified cellulose,polyethylene, polyvinyl chloride, polyvinyl acetate, copolymers of vinylacetate and vinyl chloride, polyamide, polycarbonate, polystyrene, andother polymers. Membranes formed by the classical phase inversionprocess may also be used. Thus, the non-bibulous solid supports, ingeneral, will be constructed of an inert material and will optimally beless than 1 mm in thickness and allow a capillary flow rate of <100sec/5 cm.

Bibulous materials can be converted to those which exhibit nonbibulousflow characteristics by the application of blocking agents, inparticular certain detergents and proteins, which obscure theinteractive forces that account for the bibulous nature of the supportsper se. Thus, nonbibulous solid support materials can be comprised ofbibulous materials which have been blocked. Preferred blocking agentsinclude bovine serum albumin, either per se or in methylated orsuccinylated form, whole animal sera, such as horse serum or fetal calfserum, and other blood proteins. Other protein blocking agents includecasein and non-fat dry milk.

Detergent-based blocking agents can also be used. The types ofdetergents which are appropriate are selected from nonionic, cationic,anionic and amphoteric forms, and the selection is based on the natureof the membrane being blocked. Considerations which govern the selectionof the appropriate detergent blocking agent are well understood in theart. It is preferred to use detergents in combination with protein-basedblocking agents. Suitable detergents which can be used either alone orin admixture with the protein blocking agents include polyoxyethylenesorbitan alcohol detergents (i.e., the Tween series), polyoxyethylenealcohols such as Nonidet P-40 or polyoxyethylene ethers such as TritonX-100. The selection of blocking agent and formulation of the blockingcomposition is important, as the blocking must be sufficient to effectnonbibulous flow, but the modified surface must not interfere withligand-receptor binding.

Other embodiments of non-bibulous solid support are known in the art andcan be found, for example, in Pawlak et al., International PatentApplication WO 92/12428, and Sargent et al., European Patent PublicationNo. 296 724 B1, herein incorporated by reference.

In general, the testing substrate materials are chosen such that theyenable the time to result and specificity described herein. In addition,the materials must allow the assay to proceed effectively when theliquid sample includes solution attributes that are likely to bepresent, such as milk, sugar, etc. Selection of these materials mayproceed empirically, as parameters critical to the present invention maybe found to have substantial lot-to-lot and intra-lot variability.

Alternatively, the sample application zone and the particle zone arecombined and located on material different from the rest of the solidsupport according to one embodiment. Such optional material, hereafterreferred to as an application pad, facilitates the mixing of theparticles with the liquid sample before the sample migrates through theone or more signal areas. Thus, the application pad is also in fluidflow contact with the signal areas. Fluid flow contact can includephysical contact of the application pad to the rest of the testingsubstrate, as well as the separation of the pad from the testingsubstrate by an intervening space or additional material which stillallows fluid flow between the pad and the testing substrate.Substantially all of the application pad can overlap the testingsubstrate to enable the test sample to pass through substantially anypart of the application pad to the proximal end of the testingsubstrate. Alternatively, only a portion of the application pad may bein fluid flow contact with the testing substrate. The application padcan be any material which is capable of transferring the test sample tothe testing substrate and which is able to absorb a volume of samplenecessary for obtaining an accurate and reproducible test result.

The testing substrate can have a sufficient inherent strength to be usedwithout a backing material, or additional strength can be provided bymeans of additional backing. The testing substrate can be a singlestructure such as a sheet cut into strips or it can be particulatematerial bound to a support or solid surface such as found, for example,in thin-layer chromatography.

A backing is used for support of the testing substrate in someembodiments. The backing preferably is water insoluble, non-porous, andrigid and usually will be of the same length and width as the solidsupport but can be larger or smaller. A wide variety of organic andinorganic materials, both natural and synthetic, and combinationsthereof, can be employed provided only that the backing does notinterfere with the capillary action of the strip, or non-specificallybind assay components, or interfere with the signal means. Illustrativematerials include polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), glass, ceramics, metals, and the like.

The particular dimensions of the testing substrate will be a matter ofconvenience, depending upon the size of the sample involved, the assayprotocol, the means for detecting and measuring the signal, and thelike. For example, the dimensions may be chosen to regulate the rate offluid migration as well as the amount of sample to be imbibed by poroustesting substrate.

Optionally, the testing substrate can be partially or fully enclosed ina moisture-impermeable, inert casing that can be transparent,translucent, or opaque, as known in the art. Such a casing ideally hasat least two apertures, one above the sample application zone and oneabove the signal area(s). The aperture above the signal area(s) can becovered with a transparent material. Alternatively, no apertures abovethe sample receiving zone are necessary if a bibulous means is providedto the exterior of the casing and to the testing substrate below thesample receiving zone such that the sample would be wicked in andapplied to the testing substrate. Examples of such casings can be foundin European Patent Publication No. 290 194, and as described inconjunction with FIGS. 6A-6D.

The first members of the ligand-receptor pairs may be covalently ornon-covalently bound to the particles. This binding is accomplished byany method known in the art such as, for example, the use ofglutaraldehyde and aminosilanes, as well as other methods described in“Immobilized Enzymes,” Ichiro Chibata, Halstead Press, NY (1978);Cutrecasas, J. Bio. Chem., 245:3059 (1970); March et al., Anal. Biochem,60:149, et seq. (1974); Cantarero et al., “The AbsorptionCharacteristics of Proteins for Polystyrene and Their Significance inSolid phase Immunoassays,” Analytical Biochemistry, 105:375-382 (1980);and Bangs, “Latex Immunoassays,” J. Clin. Immunoassay, 13:127-131(1980), Weng et al., and U.S. Pat. Nos. 4,740,468, 4,916,056, 3,857,931,4,181,636, and 4,264,766, each of which is incorporated herein byreference. Non-covalent binding, when used, takes advantage of thenatural adhesion of first members to the non-synthetic and especiallythe synthetic fibers. Thus, appropriately buffered solutions can bemixed with the particles then evaporated, leaving a coating of thedesired first member of the ligand-receptor pair on the particle.

The particles may be applied to the particle region of the testingsubstrate by means known in the art. Various “printing” techniques havepreviously been proposed for application of such liquid reagents tocarriers, for example, micro-syringes, pens using metered pumps, directprinting and ink-jet printing, and any of these techniques, or othertechniques that produce the same result that are yet to be discovered,can be used in the present context. To facilitate manufacture, thetesting substrate can be treated with the particles and then subdividedinto smaller portions (e.g., small, narrow strips each embodying therequired areas and regions) to provide a plurality of substantiallyidentical testing substrates.

In one embodiment, the signal generation can reflect a property of theparticles themselves. For example, the particles may themselvesintrinsically provide a detectable signal when they comprise a metalsol, a selenium sol or a carbon sol (see, e.g., U.S. Pat. Nos.4,313,734, 4,775,636, 4,954,452, and 5,559,041 each of which isincorporated by reference herein), comprise colored latex particles(e.g., as described in U.S. Pat. No. 4,703,017, incorporated byreference above) or comprise an enzyme that has reacted with a colorlesssubstrate to give a colored product and is encapsulated, for example, ina liposome. (See, e.g., International Patent Application No. WO94/01774, hereby incorporated by reference herein). Alternatively, thesignal may reflect an inducible property of the particles, such ascolorable latex particles (e.g., as described in U.S. Pat. Nos.4,373,932 and 4,837,168, each incorporated by reference above).

Alternatively the signal can be generated by a component that can beattached, either covalently or non-covalently, to either the particleitself, to one or more members of the ligand-receptor pair bound to theparticle, or both. Such component can comprise a radioisotope, such astritium, carbon 14, phosphorous 32, iodine 125, iodine 131, and thelike. Fluorescent molecules, such as the rhodamine, fluorescein, orumbelliferone series, employed by themselves or with a quenchermolecule, also can be used. (See, e.g., U.S. Pat. Nos. 3,996,345 and4,366,241, each of which are hereby incorporated by reference herein.)Chemiluminescent molecules, such as luminol, luciferin, lucigenin, oroxalyl chloride can be used as signal generating components (see, e.g.,U.S. Pat. No. 4,104,029, herein incorporated by reference). Finally,enzymic systems that react with a colorless substrate to give a coloredproduct, such as horseradish peroxidase and aminoethylcarbazole areuseful as signal generating components.

Signals detectable by visible inspection are preferred. Of these visiblesignals, those provided by colored microparticles are preferred.

The present first members can be covalently bound to radioisotopes suchas tritium, carbon 14, phosphorous 32, iodine 125 and iodine 131 bymethods well known in the art. Examples of these techniques arediscussed, e.g., in H. Van Vunakis and J. J. Langone, Editors, Methodsin Enzymology, Vol. 70, Part A (1980), and U.S. Pat. Nos. 3,646,346 and4,062,733, each of which are hereby incorporated by reference herein.Similarly, the method of conjugation and use for fluorescent moleculescan be found in the art. See, e.g, J. J. Langone, H. Van Vunkais et al.,Methods in Enzymology, Vol. 74, Part C (1981), and U.S. Pat. Nos.4,366,241, 3,996,345, and 4,104,029, each hereby incorporated byreference herein.

Enzymatic signaling components are known in the art and include singleand dual (“channeled”) enzymes such as alkaline phosphatase, horseradishperoxidase, luciferase, .beta.-galactosidase, glucose oxidase, lysozyme,malate dehydrogenase, glucose-6-phosphate dehydrogenase, and the like.Examples of channeled catalytic systems include alkaline phosphatase andglucose oxidase using glucose-6-phosphate as the initial substrate. Asecond example of such a dual enzyme system is illustrated by theoxidation of glucose to hydrogen peroxide by glucose oxidase, whichhydrogen peroxide would react with a leuco dye to produce a signalgenerator. For examples, see U.S. Pat. Nos. 4,366,241, 4,740,468,4,843,000, and 4,849,338, issued Jul. 18, 1989, each hereby incorporatedherein by reference.

The substrates for the catalytic systems include simple chromogens andfluorogens such as para-nitrophenyl phosphate (PNPP), beta-D-glucose(plus optionally a suitable redox dye), homovanillic acid,o-dianisidine, bromocresol purple powder, 4-alkyl-umbelliferone,luminol, para-dimethylaminolophine, parametholxylophine, AMPPD, and thelike. Preferred substrates for the enzymatic signal means are those thatproduce insoluble products. Examples of such preferred enzymatic signalcomponents include aminoethylcarbazole and horseradish peroxidase; andbromochloroindolyl phosphate and nitro blue tetrazolium in conjunctionwith alkaline phosphate.

The procedures for coupling enzymes to the present first members arewell known in the art and are described, for example, in J. H. Kennedyet al., Clin. Chim Acta, 70:1 (1976)). Reagents used for this procedureinclude glutaraldehyde, p-toluene diisocyanate, various carbodiimidereagents, p-benzoquinone m-periodate, N,N₁-o-phenylenedimaleimide, andthe like.

Materials preferred for use in the optional application pad includenitrocellulose, porous polyethylene filter pads and glass fiber filterpaper. The material must also be chosen for its compatibility withcaffeine and assay reagents. In addition, the optional application padcan contain one or more assay reagents either diffusively ornon-diffusively attached thereto. Reagents which can be contained in theapplication pad include buffers, preservatives, detergents,bacteriostats, ancillary ligand-receptor members, and any signal meanscomponents, such as enzyme substrates. For further discussion of such anapplication pad, see European Patent Publication No. 323 605 B1, herebyincorporated by reference herein.

The second members of the ligand-receptor pairs may be non-diffusivelybound by direct or indirect means directly to the testing substrate. Thetesting substrate may have been previously derivatized prior to theapplication of the second member. The direct binding can be covalent ornon-covalent. Covalent binding can be accomplished by using a solidsupport derivatized with one or more reactive groups such as amino,chloromethyl, aldehyde, carboxyl, epoxy, and the like. Covalent bindingcan also be accomplished by any method known in the art such as, forexample, the use of glutaraldehyde, aminosilanes, cyanogen bromide,carbonyldiimidazole, ethyl chloroformate,1-(3-nitrobenzyloxy-methyl)-pyridimium chloride (NBPC) and treslylchloride, as well as other methods, e.g., as described in “ImmobilizedEnzymes,” Ichiro Chibata, Halstead Press, NY (1978); Cutrecasas, J. Bio.Chem., 245:3059 (1970); March et al., Anal. Biochem., 60:149, et seq.(1974); and Tijssen et al., Practice and Theory of Enzyme Immunoassays,Chapter 3, Elsevier Science Publishers, (1985). The non-covalent bindingtakes advantage of the natural adhesion of second members to thenon-synthetic and especially the synthetic fibers. Thus, appropriatelybuffered solutions can be mixed with the testing substrate thenevaporated, leaving a coating of the desired second member of theligand-receptor pair on the membrane.

The non-direct method for applying the second members to the solidsupport employs either covalently or non-covalently binding the secondmembers to microparticles. Such microparticles may then be bound to orentrapped by the testing substrate such that the microparticles arewithin the matrix of the membrane, on the surface of the membrane, orbound to other microparticles which are in turn bound to the membrane.The size of the microparticles should be such that they do not migratethrough the membrane to any significant degree. The microparticles maybe made of a variety of naturally-occurring or synthetic materials, suchas microparticles are those made from polyethylene, polystyrene,agarose, dextran, cellulose, starch, or the like and the aldehyde,carboxyl, amino, hydroxyl, or hydrazide derivatives thereof. The bindingof the second member to the microparticle may be by methods similar tothose discussed above for binding the second member directly to thetesting substrate or other methods known to those skilled in the art, asdiscussed above for the preparation of the particles.

The second members, whether bound to a microparticle or not, can beapplied to the testing substrate by the means discussed above forapplying the microparticles containing the first members. In applyingthe second members to the solid support, it is necessary that theinverse signal area(s) span the width and the depth of the solvent frontcreated by any fluid traversing through the testing substrate. Suchfluid may be the sample solution, a wicking fluid as described below, ora solution containing the substrate for an enzymatic signal means. It isoptimal, but not necessary, that the direct signal areas be the samewidth as the inverse signal areas.

Antibodies, and fragments thereof, suitable for use in this inventionare obtained by techniques known to the art. For instance, polyclonalantibodies are obtained by immunizing a species of animal that differsfrom the species producing the antigen. Monoclonal antibodies areobtained by fusing the splenocytes of an immunized animal with aplasmacytoma cell line by the addition of polyethylene glycol to thecell mixture, thereby forming hybridoma cells which are suspended andthen plated to tissue culture plates. Only the cultures producingantibodies that are immunologically reactive with antigen are cloned.See, e.g., U.S. Pat. No. 4,376,110, hereby incorporated herein byreference. See also Example 1 below.

In the method, the sample can be applied to the sample application zoneas a viscous sample or a solid sample. Optionally, a wicking fluid canbe subsequently applied to sample application zone such that caffeine isdissolved or suspended in the wicking solution and the wicking solutiontraverses the particle zone and the one or more signal ratio zones inthe proper fashion. When an aqueous test sample is used, a wickingsolution generally is not necessary but can be used to improve flowcharacteristics or adjust the pH of the sample solution. In general, thewicking solution used in the present invention typically has a pH rangefrom about 5.5 to about 10.5, and more preferably from about 6.5 toabout 9.5. The pH is selected to maintain a significant level of bindingaffinity between the members of each ligand-receptor pair. When thesignal is generated using an enzyme, the pH also must be selected tomaintain significant enzyme activity for color development in enzymaticsignal production systems. Illustrative buffers include phosphate,carbonate, barbital, diethylamine, tris, 2-amino-2-methyl-1-propanol,and the like. The wicking solution and the sample can be combined priorto contacting the application pad or they can be contacted to theapplication zone sequentially.

In order to determine the ratio in the signal ratio areas, for certainof the signal generating components, it is necessary to supply separatesignal generation substance, such as a substrate for an enzyme, beforedetecting the ratio. Thus, if the signal generating component is anenzyme that converts a colorless substrate to a colored product, (e.g.,horseradish peroxidase and aminoethylcarbazole) then the substrate isapplied simultaneously with or after the application of the samplesolution.

As a matter of convenience, the present device can be provided in a kitin packaged combination with predetermined amounts of reagents for usein assaying for caffeine. Where an enzyme is used as the label, thesubstrate for the enzyme or precursors therefor including any additionalsubstrates, enzymes and cofactors and any reaction partner of theenzymic product required to provide the detectable signal can beincluded. In addition, other additives such as ancillary reagents can beincluded, for example, stabilizers, buffers, and the like. The relativeamounts of the various reagents can be varied widely, to provide forconcentrations in solution of the reagents which substantially optimizethe sensitivity of the assay. The reagents can be provided as drypowders, usually lyophilized, including excipients, which on dissolutionwill provide for a reagent solution having the appropriateconcentrations for performing the assay. The kit can also be containedin packaging material, such as air-tight foil, or various externalcontainers known in the art. Such external containers can contain thedevice, reagents, and the instructions for use of the device.

Various embodiments of casings are within the scope of the presentinvention. Examples of casings, or housings, are shown in FIG. 6A-6D.FIG. 6A is an illustration of one embodiment of a housing 605 for thetesting substrate 110 of the present invention. In this example, thehousing 605 foldable, e.g., about a hinge 610, for more compact storage.The folded housing 605 is stored further enclosed within a caseaccording to one embodiment, along with testing substrates 110. Thehousing 605 of this example includes a receptacle 615 for obtaining theliquid sample for the assay. The testing substrate 110 is placed uponthe housing 605 once the sample is obtained according to one embodiment,preferably on a flat surface. In the embodiment depicted in FIG. 6A, thereceptacle 615 is designed to allow an appropriate amount of the liquidsample to be applied to the testing substrate 110.

FIG. 6B shows another embodiment of a housing 605, in which the housingis shaped to resemble a spoon. The housing 605 shown in FIG. 6B alsoincludes a receptacle 615 for use as described above according to oneembodiment, and may also have a case. The housing 605 of FIG. 6Bincludes a transparent compartment 620 for insertion of the testingsubstrate 110, and to hold the testing substrate 110 in place during theassay according to one embodiment.

FIG. 6C shows yet another embodiment of a housing 605. In this example,the receptacle 615 of the housing 605 is dipped into the liquid samplefor collection of an appropriate amount for the assay. If the housing605 is opaque, as shown in FIG. 6C, a viewing window 625 in included sothat results of the assay may be easily seen according to oneembodiment.

FIG. 6D shows yet another embodiment of a housing 605. Like the examplein FIG. 6D, this example has a receptacle 615 that is dipped into theliquid sample for sample collection. If the housing 605 is transparent,as shown in FIG. 6D, no viewing window is required according to oneembodiment.

The invention can be better understood by way of the following exampleswhich are representative of the preferred embodiments thereof, but whichare not to be construed as limiting the scope of the invention.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of protein chemistry, biochemistry,recombinant DNA techniques and pharmacology, within the skill of theart. Such techniques are explained fully in the literature. See, e.g.,T. E. Creighton, Proteins. Structures and Molecular Properties (W. H.Freeman and Company, 1993); A. L. Lehninger, Biochemistry (WorthPublishers, Inc., current edition); Sambrook, et al., Molecular Cloning.A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S.Colowick and N. Kaplan eds., Academic Press, Inc.); Remington'sPharmaceutical Sciences, 18^(th) Edition (Easton, Pa.: Mack PublishingCompany, 1990); Carey and Sundberg Advanced Organic Chemistry 3^(rd) Ed.(Plenum Press) Vols A and B (1992).

Example 1 Development of Anti-Caffeine Antibodies

Anti-caffeine monoclonal antibodies (MAb) were produced with regard toacceptance criteria that included high-affinity binding to caffeine,minimal cross-reactivity with non-caffeine alkaloids found in coffee(e.g., theophylline, theobromine, etc.), isotypes that allow forcost-effective production of purified monoclonal antibodies, andproduction of hybridoma cell lines that secrete high levels of MAb.

To synthesize caffeine immunogens and immunize mice, sera from immunizedmice were screened for caffeine-binding antibodies by a competitiveELISA modeled on previously described protocols, e.g., as described inFickling, S. A. et al., “Development of an Enzyme-linked ImmunosorbentAssay for Caffeine,” J. Immunol. Meths., 129(2):159-64 (1990). Briefly,BSA-caffeine conjugates were synthesized using standard methods,including the linkers DSS, EMCS-IT, and others. These methods aredescribed, e.g., in Wong S S, Wong L J., “Chemical Crosslinking and theStabilization of Proteins and Enzymes,” Enzyme Microb. Technol.,14(11):866-74 (1992); Mattson, G., et al., “A Practical Approach toCrosslinking,” Molecular Biology Reports, 17, 167-183 (1993); andPartis, M. D., et al. (1983), “Crosslinking of Proteins byomega-maleimido alkanoyl N-hydroxysuccinimide Esters,” J. Protein.Chem., 2, 263-277 (1983). The BSA-caffeine conjugates were used in ELISAaccording to standard ELISA protocols, e.g., as described in Harlow &Lane. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1988. BSA-caffeine conjugate was coated ontohigh protein-binding EIA plates, incubated with serum samples with andwithout the presence of free caffeine, and antibody binding was detectedusing alkaline-phosphatase-linked goat anti-mouse-immunoglobulin (Ig)antiserum, followed by incubation with p-nitrophenyl phosphate.Substrate conversion rate was measured by optical density at 405 nm.Anti-caffeine antibodies were identified by decreased binding toBSA-caffeine when free caffeine was present.

Hybridomas producing MAbs fitting these acceptance criteria wereselected, expanded, and preserved in cryobanks. MAbs for use indevelopment of the caffeine test strips were purified from secretions ofthese hybridomas. After identification of mice with high titers ofanti-caffeine antibodies, fusions were performed to isolate hybridomassecreting anti-caffeine antibodies. Such hybridomas were identified byscreening hybridoma cell culture supernatants using the same competitiveimmunoassay.

Four hybridomas secreting anti-caffeine antibodies were identified,subcloned to generate stable cell lines, and cryopreserved. Thesehybridomas/antibodies are referenced herein as 672-1, 672-2, 672-3, and672-4. Results from competitive immunoassays of these antibodies areshown in Table 2.

TABLE 2 MAb OD, No Caffeine OD, With Caffeine 672-1 1.004 0.077 672-20.876 0.055 672-3 0.664 0.021 672-4 1.114 0.223

Table 2 shows binding of antibody-containing hybridoma supernatants toBSA-caffeine with and without the presence of free caffeine (100 ug/ml).Data is shown as mean optical density (OD) at 405 nm after subtractionof buffer-only blank.

The isotype of these antibodies was determined using a commercial kitavailable from SouthernBiotech of Birmingham, Ala. All of the antibodieswere shown to have the IgG isotype, well suited for facile purification.Furthermore, binding of antibodies to various components of theimmunochromatographic test strip has been optimized, e.g., as reviewedin Bangs, L. B., Pure & Appl. Chem., 68(10):1873-79 (1996).

If an antibody has a very high level of cross-reactivity with any ofthese alkaloids, it may be undesirable for a caffeine test kit: when asample contains very low levels of caffeine but high levels of thecross-reactant, this would lead to a false indication of caffeineconcentration. The cross-reactivity of each antibody with otheralkaloids normally contained in coffee was determined, includingtheophylline, theobromine, guanine, and adenine. The level ofcross-reactivity was determined with the same competitive ELISA, usingeach alkaloid as the competitor in place of caffeine. Data from arepresentative experiment is shown in FIG. 8 (testing of antibody672-1). None of the antibodies showed any significant cross-reactivitywith any of the tested alkaloids.

Anti-caffeine MAbs 672-1 through 672-4 were further tested for usewithin the methods described herein. The sensitivity of each antibodyfor caffeine in the competitive immunoassay was analyzed. This wasaccomplished by varying the concentration of free caffeine while theconcentration of antibody remained constant. In determinations ofreceptor binding affinity of a ligand using a competitive binding curve,the IC50 is the concentration required for 50% inhibition; antibodiescan be compared to each other by the IC50 of each antibody'sdose-response curve in a competitive assay. See descriptions ofstatistical analysis of immunoassay data in: Kurtz, D. A., et al., eds.,“New Frontiers in Agricultural Immunoassay,” AOAC International,Arlington, Va. (1995). Results of these assays are shown in Tables 3Aand 3B, and FIGS. 9A and 9B, respectively.

TABLE 3A Conc. IC50 MAb 0 1 2.25 4.5 9 18 37 75 150 300 600 (ug/ml)672-1 100 81 84.4 65.9 53.3 44.9 30.8 21.9 12.2 3.5 2.2 12 672-4 10067.2 63.7 44.9 36.3 26.8 17.1 11.1 7.6 0.2 1 4

TABLE 3B Conc. IC50 MAb 0 1 10 100 1000 10000 (ug/ml) 672-2 100 99 83.944.7 21.2 5.6 78 672-3 100 102 71.1 67 55.5 21.7 1200

Tables 3A and 3B. IC50 determination of anti-caffeine MAbs. Data shownas percent maximal binding (binding at 0 ug/ml caffeine).

As shown in FIGS. 9A and 9B, each of the four MAbs shows a distinctIC50, bracketing the stated caffeinated/decaffeinated distinctioncriterion of 62.5 ug/ml (15 mg/8 oz). Antibody 672-3 had an estimatedIC50 (78 ug/ml), closest to the distinction criterion. The antibodydesignated as 672-3 is commercially available from Silver Lake ResearchCorporation of Monrovia, Calif.

Example 2 Selection of Testing Substrate Materials

Immunochromatographic test strips were originally developed and marketedfor home pregnancy testing in the 1980's. The industry has evolved toincorporate a variety of applications, and many suppliers have developedmaterials, manufacturing modules, and other solutions for a generalizedimmunochromatographic test strip, enabling companies to develop newproducts quickly and efficiently. Off-the shelf components were combinedand adapted through industry-standard empirical testing for use in inthe methods described herein.

The generalized form of a lateral flow immunochromatographic test stripis shown in FIGS. 5A and 5B. Reviews on the theory and practice ofimmunochromatographic test strips, the function of each component, thepositioning of reagents, and other aspects have been published. See,e.g., Bangs L. B. and Meza, M., IVD Technology (1994-1995); “RapidLateral Flow Tests,” Millipore Corporation, Bedford, Mass. (2002); “TheLatex Course,” Bangs Laboratories, Inc., Fishers, Ind. (1994).

The starting point for development of the caffeine immunochromatographictest strip was the Watersafe® Pesticide Test, manufactured by SilverLake Research Corp., of Monrovia, Calif., which is a lateral flowimmunochromatographic test strip developed to detect a small moleculeanalyte in liquid samples. It was found that the components used for theWatersafe® Pesticide Test did not perform optimally when the sample wasany of several types of coffee beverages, including latte andcappuccino. These coffee matrices were slow to wick, sometimes cloggedthe membranes completely, and produced inconsistent results at any giventime point. Materials available from manufacturers of flow media forimmunochromatographic test strips were tested to determine a viablealternative.

Suppliers of materials for immunochromatographic test strip componentsprovide some basic information with regard to each version of each typeof material. Literally hundreds of versions of each component areavailable from various manufacturers worldwide. Table 4 shows the typesof materials available for each component and the quantifiableproperties of each provided by manufacturers.

TABLE 4 Example of Component material Properties Examples ofManufacturer Backing Vinyl, polyester Thickness, adhesive type,Millipore Corp., Billerica, MA; rigidity, printability G&L Precision DieCutting, Inc., San Jose, CA Binding PVDF, Porosity, thickness, flowrate, Whatman PLC, Middlesex, UK; Membrane Nitrocellulose proteinbinding capacity Sartorius AG, Goettingen, Germany; Millipore Corp.,Billerica, MA Sample Paper, Glass Density, thickness, water Whatman PLC,Middlesex, UK; Media Fiber, Foam absorbency, flow rate, air AhlstromPaper Gorup, Mt. permeability Holly Springs, VA; Millipore Corp.,Billerica, MA Reservoir Paper, foam Density, thickness, water WhatmanPLC, Middlesex, UK; Pad absorbency Ahlstrom Paper Gorup, Mt. HollySprings, VA; Millipore Corp., Billerica, MA Protective Acrylic Clarity,rigidity, adhesive type Various printing companies Cover

Table 4. Material choices for immunochromatographic test stripcomponents.

Notably, there is substantial lot-to-lot and intra-lot variation inparameters, such as flow rate, from all manufacturers. Selection ofthese materials therefore proceeded empirically, by trying severalbatches of each type of component from several manufacturers. Multiplebatches of binding membrane, sample media, and reservoir pads fromseveral manufacturers were tested for compatibility with the methodsdescribed herein. For the tested components, even when reportedporosity, flow rate, and other parameters were identical or in the samerange, different results were sometimes seen for the coffee samples.Components were tested by substitution of each version from eachmanufacturer in the appropriate position within the test striparchitecture, and by determination of the overall effect of eachsubstitution by running the assay on a variety of coffee matrices.

According to one embodiment, the test strip was comprised of anadhesive-coated vinyl backing with a thickness of 0.3 mm, anitrocellulose binding membrane with a flow rate of 90 sec./4 cm, asample media made of a glass fiber filter 0.6 cm thick with a density of260 g/m², a reservoir pad of cellulose wick, thickness of 1 mm, and awater absorption capacity of 1000 g/m², and a protective cover of aprintable acrylic cover material with a thickness of 5 mm.

Example 3 Testing of Coffee Samples

“Decaffeinated” coffee samples were ordered between the hours of 3 p.m.and 9 p.m. over the course of approximately forty days. Samples werecollected from 100 restaurants and specialty coffee retailers. Thecaffeine concentration in these samples was tested by high performanceliquid chromatography (HPLC); assays using the testing substrate fromabove were run simultaneously on the same samples. The data from thesetests are shown in Appendix A. Table 5 shows a statistical analyses ofthe results, showing the value of the caffeine assay in detectingnon-decaffeinated coffee served as decaffeinated.

TABLE 5 HPLC Data MAb Assay Mean 23.70 Count 100.00 Minimum 1.55 Correctvs. HPLC 96.00 Maximum 325.10 Percent correct 96% Count 100.00 Percentcorrect (true caff) 96.00 True Caffeinated 25 Percent correct (truedecaf) 96.00 True Caffeinated (%) 25% True Decaffeinated 75 True Decaf(%) 75%

Table 5 shows that about 25% of coffee samples served as “decaffeinated”contained caffeine above the U.S. standard for decaffeinated beverages.The data shows that the MAb assay described herein accurately identifiedboth decaffeinated and caffeinated coffees 96% of the time.

Example 4 Testing of Tea Samples

Samples of caffeinated and decaffeinated tea are tested per theparameters outlines above for coffee testing. Lipton brand Black Tea isused for the caffeinated tea samples and Twinings brand Pure Camomiletea is used for the decaffeinated tea samples. For each test, single teabags each are placed in 6 oz. Styrofoam cups, and 8 oz. of boiling (100°Celsius) water is added to each cup. The teas each are allowed to brewfor 5 min. The testing shows that the MAb assay described hereinaccurately distinguishes decaffeinated and caffeinated teas.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

1. A method of detecting the presence of caffeine in a liquid sample,comprising: applying the liquid sample to a contact region of a testingsubstrate for detecting caffeine; flowing the liquid towards a signalregion of the testing substrate spatially distinct from the contactregion; detecting in the signal region a visible signal indicating forthe liquid sample a caffeine level corresponding to a caffeinatedbeverage; and wherein the visible signal is ready for detecting withinthree minutes of applying the liquid sample.